Interpretive Summary: Obligate intracellular, tick transmitted bacterial pathogens make the remarkable transition between the mammalian and invertebrate host using a small genome. The molecular requirements for this critical transition, the understanding of which could lead to novel interventions, are largely unknown. A major obstacle in the identification of these requirements is the difficulty of the genetic manipulations in intracellular bacteria. Such manipulations are required to add fluorescent markers and knock-out specific genes in order to link genotype with phenotype. Fluorescent markers allow for easy, in vivo, real-time visualization of the pathogen, which greatly aids in the understanding of the mechanisms employed by the bacteria to survive within the host. While the techniques for these genetic manipulations have been successfully employed on a limited scale in intracellular, tick transmitted bacteria, the long-term stability within the mammalian host and the tick transmissibility of these altered organisms has not been examined. The data presented in this paper demonstrate that Anaplasma marginale, a tick borne pathogen of cattle, can stably maintain a fluorescent marker during long-term persistent infection in the immunocompetent mammalian host and throughout a complete transmission cycle in the natural tick vector, Dermacentor andersoni. These tools are necessary to understand the underpinnings of successful tick transmission and may lead to novel, broadly applicable methods to prevent tick- borne disease.

Technical Abstract:
We tested the stability of transformed Anaplasma marginale during a complete in vivo infection cycle. Similar to wild-type, the transformed A. marginale established infection in cattle, a natural reservoir host, and persisted in immune competent animals. The tick infection rate was indistinguishable from wild-type however there were significant lower levels of the transformed A. marginale in the tick. Despite the lower level of replication, ticks transmitted the transformant. Transformants can serve as valuable tools to dissect the molecular requirements of tick colonization and pathogen transmission.